Abstract

Background

Breast cancer is one of the leading cause of cancer-related deaths in women worldwide and increasing rapidly in developing countries. In the present study, we investigated the potential role and association of HSP70-2 with breast cancer.

Methods

HSP70-2 expression was examined in 154 tumor and 103 adjacent non-cancerous tissue (ANCT) specimens and breast cancer cell lines (MCF7, BT-474, SK-BR-3 and MDA-MB-231) by RT-PCR, quantitative-PCR, immunohistochemistry, Western blotting, flow cytometry and indirect immunofluorescence. Plasmid driven short hairpin RNA approach was employed to validate the role of HSP70-2 in cellular proliferation, senescence, migration, invasion and tumor growth. Further, we studied the effect of HSP70-2 protein ablation on signaling cascades involved in apoptosis, cell cycle and Epithelial-Mesenchymal-Transition both in culture as well as in-vivo human breast xenograft mouse model.

Results

HSP70-2 expression was detected in majority of breast cancer patients (83 %) irrespective of various histotypes, stages and grades. HSP70-2 expression was also observed in all breast cancer cells (BT-474, MCF7, MDA-MB-231 and SK-BR-3) used in this study. Depletion of HSP70-2 in MDA-MB-231 and MCF7 cells resulted in a significant reduction in cellular growth, motility, onset of apoptosis, senescence, cell cycle arrest as well as reduction of tumor growth in the xenograft model. At molecular level, down-regulation of HSP70-2 resulted in reduced expression of cyclins, cyclin dependent kinases, anti-apoptotic molecules and mesenchymal markers and enhanced expression of CDK inhibitors, caspases, pro-apoptotic molecules and epithelial markers.

Conclusions

HSP70-2 is over expressed in breast cancer patients and was involved in malignant properties of breast cancer. This suggests HSP70-2 may be potential candidate molecule for development of better breast cancer treatment.

Keywords

HSP70-2Breast cancerGene silencingApoptosisTumor growth

Background

Breast cancer is the second leading cause of cancer related deaths among women worldwide [1]. Breast cancers are mostly adenocarcinomas which include invasive type such as infiltrating ductal carcinoma (IDC) and non-invasive type, Ductal Carcinoma in-situ (DCIS) [2]. IDC is the most common subtype of breast cancer accounting for 75–80 % of all the cases diagnosed [3]. Breast cancer incidence is highest in developed countries and increasing rapidly in developing countries due to lack of adequate medical support and infrastructure [4]. Therefore, there is a need to characterize a tumor-associated molecule for early detection of breast cancer and for identifying a novel therapeutic target for better cancer treatment.

Heat shock family of proteins (HSPs) is proposed to play pivotal role in breast tumor development owing to their intrinsic molecular chaperone properties [5]. HSPs are mainly involved in protein folding, assembly of multiprotein complexes, protein transport and protein targeting for lysosomal degradation [5]. Few of the HSPs have been reported in breast cancer which includes HSP27, HSP90 and HSP70 [6–8]. HSP70 family has eight members with high homology but different cellular localization and biological functions [8]. HSP70-2, a new member of cancer testis (CT) antigen family and an also an important member of HSP70 family has been found to be associated with various malignancies [8–11]. However, the clinical relevance and association of HSP70-2 expression in IDC specimens have not been investigated so far.

Our earlier studies have shown a close association of HSP70-2 with cellular proliferation, migration, invasion and tumor growth in urothelial [9] and cervical cancer [10]. In the present study, we have investigated the possible association of HSP70-2 mRNA and protein expression with various stages, grades and histotypes of breast cancer patients. In addition, we have examined the putative role of HSP70-2 in apoptosis, cell cycle arrest and epithelial-mesenchymal transition (EMT) in-vitro and in-vivo xenograft mouse model using gene silencing approach. Here we provide evidence that HSP70-2 expression is associated with IDC histotype of breast cancer. We also report that HSP70-2 plays an important role in cellular growth, migration and invasion of breast cancer cells and tumor growth of breast cancer xenograft. At molecular level, we show that HSP70-2 depletion resulted in up-regulation of caspases, pro-apoptotic molecules, cyclin dependent kinase (CDK) inhibitors and epithelial markers, and down-regulation of anti-apoptotic molecules, cyclins, CDKs and mesenchymal markers. These results collectively suggest that HSP70-2 could be used as a candidate for developing a novel therapeutic in breast cancer management.

Methods

Patient samples

The tumor specimens from 154 patients and 103 matched available adjacent non-cancerous tissue (ANCT) specimens were collected during surgical procedure at All India Institute of medical Sciences (AIIMS), New Delhi; Mahatma Gandhi Medical Hospital, Jaipur. All tumor specimens were collected from new patients who had not undergone treatment before surgery (study period 2008–2014). Details of specimens are illustrated in Table 1. The study was conducted as per the Ethical Committee approval obtained from AIIMS, Mahatma Gandhi Medical Hospital, Jaipur and National Institute of Immunology, New Delhi. The duly signed consent forms were obtained from the patients prior to the study.

HSP70-2 mRNA and protein expression was examined in tumor specimens and all four breast cancer cell lines along with HNMEC’s as detailed in Additional file 1 and Methods section.

Immunoreactivity score (IRS)

Immunoreactivity score (IRS) was calculated as a percentage of cells expressing HSP70-2 protein. For determining the IRS, the tissue section slides were independently reviewed by two senior pathologists. More than 500 cells were counted from five random fields at 400× magnifications. Specimens showing >10 % HSP70-2 positive cells were considered as positive immuno-reactive specimens.

Validation of shRNA targets against HSP70-2

Four shRNA constructs against HSP70-2 along with scrambled negative control NC shRNA were procured from Super Array (Frederick, MD, USA) as detailed earlier [9]. The transient transfections were carried out in MCF7 and MDA-MB-231 cells using lipofectamine (Invitrogen Life Technologies Corporation, USA) and HSP70-2 knockdown efficiency was determined by Real-time PCR and Western blotting as described in Additional file 1 and Methods section.

Cell cycle analysis

The HSP70-2 shRNA3, shRNA4 and NC shRNA transfected MDA-MB-231 cells were fixed in ethanol and stained with propidium iodide with RNaseA as described earlier [12]. The cells were analyzed by flow cytometry on BD-VERSA (BD Biosciences, California, USA).

AnnexinV staining

To study the effect of HSP70-2 shRNA3 and shRNA4 on apoptosis compared to NC shRNA, MDA-MB-231 cells were stained with annexinV using annexinV-PerCP-Cy5-5-A staining kit (Biovision, CA, USA) and assay carried out as described earlier [13].

Cell migration, invasion and wound healing assay

Cell migration, invasion and wound healing ability of HSP70-2 shRNA3, shRNA4 and NC shRNA transfected MCF7 and MDA-MB-231 cells were assessed as described in Additional file 1 and Methods section.

In-vivo xenograft studies

Athymic nude mice (National Institute of Immunology [NII], National Institutes of Health, [S] nu/nu) were used for the xenograft studies. Mice were injected subcutaneously with 5 × 106 MDA-MB-231 cells. The experiment was conducted as described in Additional file 1 and Methods section.

Statistical analysis

The statistical analysis was performed using SPSS 20.0 statistical software package (SPSS Inc, Chicago, IL, USA). Pearson’s Chi-Square test was performed among various grades and stages. P-values ≤ 0.017 among grades and P-value ≤ 0.013 were considered as statistically significant after applying a Bonferroni correction for multiple comparisons. Based on immuno-reactivity score (IRS), the IDC specimens were divided in; Group I including specimens with >50 % cells expressing HSP70-2 protein, Group II; including specimens with <50 % cells expressing this protein. The statistical difference of HSP70-2 protein expressing cells in group I and group II was determined by Mann Whitney test. Statistical comparisons of mean values in cell line study were performed using Student’s t-test. A P-value <0.05 was considered statistically significant. Data is expressed as mean ± standard error of three independent experiments in triplicates in in-vitro assays.

HSP70-2 is over-expressed in breast cancer cell lines

Since breast tumor samples showed overexpression of HSP70-2 mRNA as well as protein, we next examined its expression in four different breast cancer cell lines viz., BT-474, MCF7, MDA-MB-231 and SK-BR-3. As shown in Fig. 1c, HSP70-2 mRNA expression was detected in all four breast cancer cells irrespective of their molecular phenotype but not in human normal mammary epithelial cells (HNMEC; Fig. 1c). There was higher HSP70-2 mRNA expression in triple negative MDA-MB-231 (>7 fold; P < 0.001) cells, followed by MCF7 (>3fold; P < 0.001), SK-BR-3 (>3 fold; P < 0.001) and BT-474 (1.28 fold; P < 0.05) with respect to HNMEC (Fig. 1d). Further Western blot analysis confirmed the expression of HSP70-2 protein in in all cell lines (Fig. 1e) along with their surface localization (Fig. 1f). In addition, we also observed cytoplasmic presence of HSP70-2 in all these cells (Additional file 2: Figure S1e) with specific localization in endoplasmic reticulum, mitochondria and Golgi bodies but not with nuclear envelope (Fig. 1g).

HSP70-2 gene silencing initiates apoptosis

The phenotypic changes associated with apoptosis were investigated by SEM in HSP70-2 depleted MCF7 and MDA-MB-231 cells. Post 24 h, both breast cancer cells exhibited early signs of apoptosis including cell shrinkage and blebbing, followed by the formation of apoptotic bodies in HSP70-2 shRNA3 and shRNA4 transfected cells (Fig. 3a). Post 48 h, most of the cells appeared to undergo apoptotic death. However, no phenotypic changes in the morphology of NC shRNA transfected (Fig. 3a) and lipofectamine treated cells were observed (Additional file 4: Figure S3a).

To elucidate the role of HSP70-2 in apoptosis, we analyzed several characteristic markers of apoptosis in HSP70-2 depleted cells. We analyzed annexinV staining to examine the externalization of phosphatidyl serine, an early sign of apoptosis. Our flow cytometry data showed marked increase in annexinV staining in MDA-MB-231 cells transfected with HSP70-2 shRNA3 and shRNA4 as compared to NC shRNA (Fig. 3b). We also determined changes in the mitochondrial membrane potential (MMP) of HSP70-2 depleted MDA-MB-231 cells using TMRE (tetramethyl rhodamine, ethyl ester) dye. Our data revealed loss of potential due to HSP70-2 shRNA treatment (Fig. 3c). Next, we investigated caspase cleavage by employing M30 assay and found higher population of M30 positive (Fig. 3d). To monitor the late apoptosis events, we also examined chromatin condensation by DAPI staining and observed a rise in the DAPI intensity in HSP70-2 depleted MDA-MB-231 cells (Fig. 3e). In addition, we examined DNA fragmentation by TUNEL assay and observed a marked increase in BrdU positive MDA-MB-231cells (Fig. 3f).

Collectively, our results indicated that HSP70-2 contributes in cellular proliferation, cellular migration and invasion and its depletion causes significant reduction in these processes both in-vitro and in-vivo xenograft mouse model.

Discussion

Breast cancer is the most common cancer in women worldwide and is the second leading cause of cancer-related death in women [1]. Among various histotypes of breast cancer, infiltrating ductal carcinoma (IDC) is the most common histotype of breast cancer in which cancer cells invade through the ductal wall into the stroma [2]. Therefore, the present study was undertaken to investigate the role of a novel cancer testis (CT) antigen, HSP70-2 expression and its potential involvement in breast cancer patients and in various breast cancer cell line models. We found that majority of breast cancer patients [83 % (128/154), Table 1] showed HSP70-2 expression irrespective of stages, grades and histotypes. More importantly, we also found significant association of HSP70-2 expression with grades. In addition, HSP70-2 expression was observed in majority of patients with positive lymph node involvement suggesting a role of HSP70-2 in tumor migration and invasion. Our data from various breast cancer cell lines were broadly in agreement with our in-vivo studies in xenograft model. Earlier studies in small number of breast cancer specimens [36 % (9/25)] also revealed HSP70-2 expression [8]. Moreover our recent studies on well characterized CT antigens, SPAG9 and AKAP4 have shown their association with breast cancer cases [14, 15]. The other CT antigens family member, MAGE-A9 and MAGE-A11 have also been found to be expressed in breast cancer specimens [16]. Our current findings in various stages and grades of breast cancer tumors suggest that HSP70-2 may be involved in various signaling pathways and control cellular proliferation, dysregulation of cell cycle, migration and invasion abilities in cancer cells.

Altered expression of cell-cycle-regulatory proteins is a major abnormalities during cancer [17]. It may involve over-expression of cyclins and cyclin dependent kinases (CDK) or mutation of tumor suppressor genes [18]. A earlier report has shown that HSP70-2 is a binding partner of cdk1/cyclinB complex during meiotic division in spermatogenesis [19]. The present study shows that ablation of HSP70-2 resulted in accumulation of cancer cells in G0/G1 stage. In support of our observation, similar findings have been reported in HSP70-2 depleted HeLa cells [8]. Our results further revealed that molecules involved in cell cycle (cyclins: cyclin A2, cyclin B1, cyclin D1 and cyclin E along with CDKs: CDK1, CDK2, CDK4 and CDK6) were downregulated in HSP70-2 depleted MDA-MB-231 cells. Interestingly, we also found that senescence was associated with G0/G1 arrest with increased expression of p21, Rb and DCR2 (senescence marker). In this context, it may be possible that the HSP70-2 expression may play an important role in alteration of cell cycle regulation molecules resulting in uncontrolled cellular proliferation.

Failure in process of apoptosis results in cancer disease [17]. The cell death occurs due to initiation of apoptosis which includes well regulated expression of pro- and anti-apoptotic molecules of the Bcl-2 family as regulatory proteins [17]. Over-expression of BCL-2 has been found in 60–80 % of breast carcinoma suggesting its role in breast cancer [20]. The current study showed significant increase in cell death in HSP70-2 depleted MDA-MB-231 and MCF7 cells. Our observations further supported that cancer cell death occurred due to decreased expression in anti-apoptotic molecules (BCL-xL, BCL2, Survivin, XIAP2, cIAP2, MCL1) and increased expression in pro-apoptotic molecules (caspase 3, caspase 6, caspase 7, caspase 9, BAX, BAK, BAD, NOXA, BID, cytochrome-C, PUMA, APAF1, AIF, and PARP1) in HSP70-2 depleted MDA-MB-231 cells. Similar findings on BORIS-specific siRNA treatment induced caspase 3/7 activation in a dose dependent manner in MDA-MB-231 cells [21] lead to cell death indicating its role in survival of cancer cells. Interestingly, recent studies have shown the role of yet another important molecule, unfolded protein response (UPR) which is upregulated in triple negative breast cancer (TNBC) [22]. Interestingly, our report on HSP70-2 ablated in TNBC, MDA-MB-231 cells lead to decrease expression of glucose-regulated protein (GRP78), which is a master regulator of UPR and has been associated with cancer disease and drug resistance [23]. Collectively, our study indicates that ablation of HSP70-2 in breast cancer cells promotes apoptosis and hence cancer cell death.

Our most important findings suggest that HSP70-2 expression in breast cancer cells play an important role in cellular growth, cell migration, and invasion. Epithelial-Mesenchymal transition (EMT) is considered to be the important pathway involved in migration and invasion of cancer cells to distant sites [17]. Expression of proteins characteristic of mesenchymal cells (N-Cadherin, Vimentin, SNAIL, SLUG) and loss of epithelial markers (E-Cadherin) correlates with tumor progression and poor prognosis [24]. Our observations supported previous studies [24] wherein HSP70-2 ablation in the present study effected cancer cell migration and invasion potential in MDA-MB-231 and MCF7 cells. Supported by previous findings [24], our data also revealed that various mesenchymal markers like N-Cadherin, P-Cadherin, MMP2, MMP3, MMP9, SLUG, TWIST, Vimentin, and SMA were down-regulated with increased expression of E-Cadherin in HSP70-2 depleted MDA-MB-231 cells. Recent studies on CT antigens such as SSX, MAGED4B, CAGE and piwil2 showed up-regulation of EMT and metastatic genes expression that promote tumor dissemination [25]. A recent study also showed that over-expression of CT45A1, CT antigen in breast cancer cells selectively enhanced the expression of pro-EMT gene, including TWIST1, ALDH1A1 [26]. On similar lines, our data suggests that HSP70-2 depletion in breast cancer cells contributes in reduced cancer cell motility by inhibiting molecules involved in EMT pathway.

In summary, this investigation of well characterized human breast cancer tissues (103 ANCT and n = 154 cancer tissue) has documented that HSP70-2 expression is over expressed in breast cancer patients. Furthermore, our data demonstrated that plasmid-mediated RNA interference of HSP70-2 successfully inhibited the expression of HSP70-2 in in-vitro and in-vivo models of breast cancer, leading to inhibitory effects on cell proliferation, migration, invasion, and tumor growth. In this regard, recent, clinical trials employing gene silencing approach has shown a promise in developing new class of therapeutics [27]. Collectively, our findings suggest that the shRNA mediated gene silencing approach may be an effective therapeutic strategy as an adjuvant therapy or in combination with other treatment modalities for breast cancer and therefore warrant future investigations in human clinical trials.

Conclusions

Taken together, our study indicated that HSP70-2 might be playing an important role in development and progression of breast cancer. Gene silencing approach indicated that HSP70-2 promotes cell growth and cellular motility of breast cancer cells. Also, HSP70-2 depletion led to reduction in tumor growth in in-vivo human breast cancer xenograft model. Recent clinical trials on siRNA based therapy [27] indicated that gene silencing approach has a potential to be employed in cancer therapy. Thus, HSP70-2 may be considered as potential candidate molecule in the management of breast cancer patients and warrants future studies.

Declarations

Acknowledgements

We acknowledge Dr V. Kumar, Senior Staff Scientist, International Centre for Genetic Engineering and Biotechnology, New Delhi, India for critical reading and editing of this manuscript. We also thank technical support by Mrs. Rekha Rani, National Institute of Immunology, New Delhi, India for SEM imaging.

Funding

This work is supported by grants from Indo-UK Cancer Research Program (Grant No. BT/IN/UK/NII/2006), Centre for Molecular Medicine (Grant No. BT/PR/14549/MED/14/1291), NII-core funding, Department of Biotechnology, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

Author’ contributions

NJ, SA, NG, RF, SD and VK carried out all the experiments, prepared figures and drafted the manuscript. NJ, ASA and NKL participated in data analysis and interpretation of results. AG and VaS pathologists performed histopathology examination of all the clinical specimens used in this investigation. RK and TCS senior surgeon provided clinical samples and ViS collected clinicopathological data from the hospital for this study. AS designed the study, participated in data analysis and interpretation of results. All authors read and approved the manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Consent has been obtained from patients to publish.

Ethics approval and consent to participate

We have taken the approval from the ethics committees of the participating institutes and the consent forms were obtained from the patients. This information is included in Methods section.

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